SARS-CoV-2 infection modifies the transcriptome of the megakaryocytes in the bone marrow.

Megakaryocytes, integral to platelet production, predominantly reside in the bone marrow and undergo regulated fragmentation within sinusoid vessels to release platelets into the bloodstream. Inflammatory states and infections influence megakaryocyte transcription, potentially affecting platelet functionality. Notably, COVID-19 has been associated with altered platelet transcriptomes. In this study, we investigated the hypothesis that SARS-CoV-2 infection could impact the transcriptome of bone marrow megakaryocytes. Utilizing spatial transcriptomics to discriminate subpopulations of megakaryocytes based on proximity to bone marrow sinusoids, we identified approximately 19,000 genes in megakaryocytes. Machine learning techniques revealed that the transcriptome of healthy murine bone marrow megakaryocytes exhibited minimal differences based on proximity to sinusoid vessels. Further, at peak SARS-CoV-2 viremia, when the disease primarily affected the lungs, megakaryocytes were not significantly different from those from healthy mice. Conversely, a significant divergence in the megakaryocyte transcriptome was observed during systemic inflammation, although SARS-CoV-2 RNA was never detected in bone marrow and it was no longer detectable in the lungs. Under these conditions, the megakaryocyte transcriptional landscape was enriched in pathways associated with histone modifications, megakaryocyte differentiation, NETosis, and autoimmunity, which could not be explained by cell proximity to sinusoid vessels. Notably, the type-I interferon signature and calprotectin (S100A8/A9) were not induced in megakaryocytes under any condition. However, inflammatory cytokines induced in the blood and lungs of COVID-19 mice were different from those found in the bone marrow, suggesting a discriminating impact of inflammation on this specific subset of cells. Collectively, our data indicate that a new population of bone marrow megakaryocytes may emerge through COVID-19-related pathogenesis.

Immunoregulatory molecule expression on extracellular microvesicles in people living with HIV.

Introduction: People living with HIV (PLWH) now benefit from combined antiviral treatments that durably control viral replication. These antiretroviral treatments decrease mortality and improve quality of life in PLWH, but do not completely control the excessive non-specific activation of the immune system in PLWH. This chronic immune activation is a key element of HIV immunopathology that contributes to the pathophysiology of inflammatory comorbid conditions, such as cardiovascular disorders, cancer and autoimmune diseases. Circulating non-exosomal extracellular vesicles, also known as microparticles (MPs) are detected in these diseases and have been linked to immune activation. The objective of this study was to characterize the MPs present in PLWH and to assess their association with chronic immune activation. Methods: We performed flow cytometry for the complete phenotypic characterization of MPs from fresh plasma from PLWH and from people without HIV as the control group. The absolute number, size and cellular origin of MPs were evaluated. The immunoregulatory profile was determined by cell origin, for MPs derived from platelets (PMPs), monocytes (MMPs) and T lymphocytes (LMPs). Results: PLWH had significantly more circulating MPs than controls, for MPs of all sizes originating from T lymphocytes, red blood cells, neutrophils, dendritic cells, B lymphocytes and endothelial cells. PMPs and MMPs were not more numerous in PLWH, but the immunoregulatory phenotypes of these MPs differed between PLWH and controls. These differences in immunoregulatory molecule expression profile were also observed for LMPs. PDL1, ICOSL, CCR5, TGFß1, MHC classes I and II, TRAIL, CXCR4, OX40, DC-SIGN, CTLA4 and PDL2 were more strongly expressed on the surface of MPs from PLWH than on those from controls. Conclusion: MPs are an important element in intercellular communication, making it possible to transfer phenotypes and functions to immune cells. The significantly higher numbers of MPs expressing diverse immunomodulatory molecules in PLWH may make a major contribution to the maintenance and/or the development of immune-cell activation in these individuals.

IgG hexamers initiate complement-dependent acute lung injury.

Antibodies can initiate lung injury in a variety of disease states such as autoimmunity, transfusion reactions, or after organ transplantation, but the key factors determining in vivo pathogenicity of injury-inducing antibodies are unclear. Harmful antibodies often activate the complement cascade. A model for how IgG antibodies trigger complement activation involves interactions between IgG Fc domains driving assembly of IgG hexamer structures that activate C1 complexes. The importance of IgG hexamers in initiating injury responses was unclear, so we tested their relevance in a mouse model of alloantibody and complement-mediated acute lung injury. We used three approaches to block alloantibody hexamerization (antibody carbamylation, the K439E Fc mutation, or treatment with domain B from Staphylococcal protein A), all of which reduced acute lung injury. Conversely, Fc mutations promoting spontaneous hexamerization made a harmful alloantibody into a more potent inducer of acute lung injury and rendered an innocuous alloantibody pathogenic. Treatment with a recombinant Fc hexamer 'decoy' therapeutic protected mice from lung injury, including in a model with transgenic human FCGR2A expression that exacerbated pathology. These results indicate an in vivo role of IgG hexamerization in initiating acute lung injury and the potential for therapeutics that inhibit or mimic hexamerization to treat antibody-mediated diseases.

IgG hexamers initiate acute lung injury.

Antibodies can initiate lung injury in a variety of disease states such as autoimmunity, transfusion reactions, or after organ transplantation, but the key factors determining in vivo pathogenicity of injury-inducing antibodies are unclear. A previously overlooked step in complement activation by IgG antibodies has been elucidated involving interactions between IgG Fc domains that enable assembly of IgG hexamers, which can optimally activate the complement cascade. Here, we tested the in vivo relevance of IgG hexamers in a complement-dependent alloantibody model of acute lung injury. We used three approaches to block alloantibody hexamerization (antibody carbamylation, the K439E Fc mutation, or treatment with domain B from Staphylococcal protein A), all of which reduced acute lung injury. Conversely, Fc mutations promoting spontaneous hexamerization made a harmful alloantibody into a more potent inducer of acute lung injury and rendered an innocuous alloantibody pathogenic. Treatment with a recombinant Fc hexamer 'decoy' therapeutic protected mice from lung injury, including in a model with transgenic human FCGR2A expression that exacerbated pathology. These results indicate a direct in vivo role of IgG hexamerization in initiating acute lung injury and the potential for therapeutics that inhibit or mimic hexamerization to treat antibody-mediated diseases.

MIBlood-EV: Minimal information to enhance the quality and reproducibility of blood extracellular vesicle research.

Blood is the most commonly used body fluid for extracellular vesicle (EV) research. The composition of a blood sample and its derivatives (i.e., plasma and serum) are not only donor-dependent but also influenced by collection and preparation protocols. Since there are hundreds of pre-analytical protocols and over forty variables, the development of standard operating procedures for EV research is very challenging. To improve the reproducibility of blood EV research, the International Society for Extracellular Vesicles (ISEV) Blood EV Task Force proposes standardized reporting of (i) the applied blood collection and preparation protocol and (ii) the quality of the prepared plasma and serum samples. Gathering detailed information will provide insight into the performance of the protocols and more effectively identify potential confounders in the prepared plasma and serum samples. To collect this information, the ISEV Blood EV Task Force created the Minimal Information for Blood EV research (MIBlood-EV), a tool to record and report information about pre-analytical protocols used for plasma and serum preparation as well as assays used to assess the quality of these preparations. This tool does not require modifications of established local pre-analytical protocols and can be easily implemented to enhance existing databases thereby enabling evidence-based optimization of pre-analytical protocols through meta-analysis. Taken together, insight into the quality of prepared plasma and serum samples will (i) improve the quality of biobanks for EV research, (ii) guide the exchange of plasma and serum samples between biobanks and laboratories, (iii) facilitate inter-laboratory comparative EV studies, and (iv) improve the peer review process.

Diversity of Megakaryocytes.

Megakaryocytes are commonly known as large, polyploid, bone marrow resident cells that contribute to hemostasis through the production of platelets. Soon after their discovery in the 19th century, megakaryocytes were described in tissue locations other than the bone marrow, specifically in the lungs and the blood circulation. However, the localization of megakaryocytes in the lungs and the contribution of lung megakaryocytes to the general platelet pool has only recently been appreciated. Moreover, the conception of megakaryocytes as uniform cells with the sole purpose of platelet production has been challenged. Here, we review the literature on megakaryocyte cell identity and location with a special focus on recent observations of megakaryocyte subpopulations identified by transcriptomic analyses.

Platelet extracellular vesicles and their mitochondrial content improve the mitochondrial bioenergetics of cellular immune recipients.

BACKGROUND: Mitochondria play a critical role in the production of cell energy and the regulation of cell death. Therefore, mitochondria orchestrate numerous cell effector functions, including fine-tuning the immune system. While mitochondria are mainly found intracellularly, they can escape the confine of the cell during the process of extracellular vesicle release. Platelets patrol blood vessels to ensure vasculature integrity and to support the immune system. In blood, platelets are the primary source of circulating mitochondria. Activated platelets produce extracellular vesicles, including a subset of mitochondria-containing vesicles. STUDY DESIGN AND METHODS: We characterized mitochondrial functions in platelet-derived extracellular vesicles, and examined whether they could impact the bioenergetics of cellular immune recipients using an extracellular flux analyzer to measure real-time bioenergetics. RESULTS: We validated that extracellular vesicles derived from activated platelets contain the necessary mitochondrial machinery to respirate and generate energy. Moreover, neutrophils and monocytes efficiently captured platelet-derived extracellular vesicles, enhancing their mitochondrial fitness. This process required functional mitochondria from donor platelets, as it was abolished by the inactivation of extracellular mitochondria using mitochondrial poison. DISCUSSION: Together, the data suggest that extracellular mitochondria produced by platelets may support other metabolic functions through transcellular bioenergetics.

Apoptotic exosome-like vesicles transfer specific and functional mRNAs to endothelial cells by phosphatidylserine-dependent macropinocytosis.

Apoptosis of endothelial cells prompts the release of apoptotic exosome-like vesicles (ApoExos), subtype extracellular vesicles secreted by apoptotic cells after caspase-3 activation. ApoExos are different from both apoptotic bodies and classical exosomes in their protein and nucleic acid contents and functions. In contrast to classical apoptotic bodies, ApoExos induce immunogenic responses that can be maladaptive when not tightly regulated. In the present study, we elucidated the mechanisms by which ApoExos are internalized by endothelial cells, which leads to shared specific and functional mRNAs of importance to endothelial function. Using flow cytometry and confocal microscopy, we revealed that ApoExos were actively internalized by endothelial cells. SiRNA-induced inhibition of classical endocytosis pathways with pharmacological inhibitors showed that ApoExos were internalized via phosphatidylserine-dependent macropinocytosis independently of classical endocytosis pathways. An electron microscopy analysis revealed that ApoExos increased the macropinocytosis rate in endothelial cells, setting in motion a positive feedback loop that increased the amount of internalized ApoExos. Deep sequencing of total RNA revealed that ApoExos possessed a unique protein-coding RNA profile, with PCSK5 being the most abundant mRNA. Internalization of ApoExos by cells led to the transfer of this RNA content from the ApoExos to cells. Specifically, PCSK5 mRNA was transferred to cells that had taken up ApoExos, and these cells subsequently expressed PCSK5. Collectively, our findings suggest that macropinocytosis is an effective entry pathway for the delivery of RNAs carried by ApoExos and that these RNAs are functionally expressed by the endothelial cells that internalize them. As ApoExos express a specific mRNA signature, these results suggest new avenues to understand how ApoExos produced at sites of vascular injury impact vascular function.

Thermoneutrality and severe malaria: investigating the effect of warmer environmental temperatures on the inflammatory response and disease progression.

Introduction: Most studies using murine disease models are conducted at housing temperatures (20 - 22°C) that are sub-optimal (ST) for mice, eliciting changes in metabolism and response to disease. Experiments performed at a thermoneutral temperature (TT; 28 - 31°C) have revealed an altered immune response to pathogens and experimental treatments in murine disease model that have implications for their translation to clinical research. How such conditions affect the inflammatory response to infection with Plasmodium berghei ANKA (PbA) and disease progression is unknown. We hypothesized that changes in environmental temperature modulate immune cells and modify host response to malaria disease. To test this hypothesis, we conducted experiments to determine: (1) the inflammatory response to malarial agents injection in a peritonitis model and (2) disease progression in PbA-infected mice at TT compared to ST. Methods: In one study, acclimatized mice were injected intraperitoneally with native hemozoin (nHZ) or Leishmania at TT (28 - 31°C) or ST, and immune cells, cytokine, and extracellular vesicle (EV) profiles were determined from the peritoneal cavity (PEC) fluid. In another study, PbA-infected mice were monitored until end-point (i.e. experimental malaria score ≥4). Results: We found that Leishmania injection resulted in decreased cell recruitment and higher phagocytosis of nHZ in mice housed at TT. We found 398 upregulated and 293 downregulated proinflammatory genes in mice injected with nHZ, at both temperatures. We report the presence of host-derived EVs never reported before in a murine parasitic murine model at both temperatures. We observed metabolic changes in mice housed at TT, but these did not result to noticeable changes in disease progression compared to ST. Discussion: To our knowledge, these experiments are the first to investigate the effect of thermoneutrality on a malaria murine model. We found important metabolic difference in mice housed at TT. Our results offer insights on how thermoneutrality might impact a severe malaria murine model and directions for more targeted investigations.

Bioactive lipids as biomarkers of adverse reactions associated with apheresis platelet concentrate transfusion.

Platelet concentrate (PC) transfusion seeks to provide haemostasis in patients presenting severe central thrombocytopenia or severe bleeding. PCs may induce adverse reactions (AR) that can occasionally be severe (SAR). PCs contain active biomolecules such as cytokines and lipid mediators. The processing and storage of PCs creates so-called structural and biochemical storage lesions that accumulate when blood products reach their shelf life. We sought to investigate lipid mediators as bioactive molecules of interest during storage and review associations with adverse reactions post-transfusion. To facilitate understanding, we focused on single donor apheresis (SDA) PCs with approximately 31.8% of PCs being delivered in our setting. Indeed, pooled PCs are the most widely transfused products, but the study of a single donor lipid mediator is easier to interpret. We are investigating key lipid mediators involved in AR. Adverse reactions were closely monitored in accordance with current national and regional haemovigilance protocols. Residual PCs were analysed post-transfusion in a series of observations, both with and without severe reactions in recipients. A decrease in the lysophosphatidylcholine species to produce the lysophosphatidic acid species has been observed during storage and in the case of AR. Lysophosphatidic acid increased with primarily platelet-inhibitor lipids. Anti-inflammatory platelet-induced inhibition lipids were weakly expressed in cases of severe adverse reactions. We therefore propose that a decrease in lysophosphatidylcholine and an increase in lysophosphatidic acid can prospectively predict serious adverse transfusion reactions.

Bacterial extracellular vesicles and their interplay with the immune system.

The mammalian intestinal tract harbors trillions of microorganisms confined within this space by mucosal barriers. Despite these barriers, bacterial components may still be found elsewhere in the body, even in healthy subjects. Bacteria can release small lipid-bound particles, also named bacterial extracellular vesicles (bEV). While bacteria themselves cannot normally penetrate the mucosal defense, bEVs may infiltrate the barrier and disseminate throughout the body. The extremely diverse cargo that bEVs can carry, depending on their parent species, strain, and growth conditions, grant them an equally broad potential to interact with host cells and influence immune functions. Herein, we review the current knowledge of processes underlying the uptake of bEVs by mammalian cells, and their effect on the immune system. Furthermore, we discuss how bEVs could be targeted and manipulated for diverse therapeutic purposes.

SARS-CoV-2 Nsp2 Contributes to Inflammation by Activating NF-κB.

COVID-19 is associated with robust inflammation and partially impaired antiviral responses. The modulation of inflammatory gene expression by SARS-CoV-2 is not completely understood. In this study, we characterized the inflammatory and antiviral responses mounted during SARS-CoV-2 infection. K18-hACE2 mice were infected with a Wuhan-like strain of SARS-CoV-2, and the transcriptional and translational expression interferons (IFNs), cytokines, and chemokines were analyzed in mouse lung homogenates. Our results show that the infection of mice with SARS-CoV-2 induces the expression of several pro-inflammatory CC and CXC chemokines activated through NF-κB but weakly IL1ß and IL18 whose expression are more characteristic of inflammasome formation. We also observed the downregulation of several inflammasome effectors. The modulation of innate response, following expressions of non-structural protein 2 (Nsp2) and SARS-CoV-2 infection, was assessed by measuring IFNß expression and NF-κB modulation in human pulmonary cells. A robust activation of the NF-κB p65 subunit was induced following the infection of human cells with the corresponding NF-κB-driven inflammatory signature. We identified that Nsp2 expression induced the activation of the IFNß promoter through its NF-κB regulatory domain as well as activation of p65 subunit phosphorylation. The present studies suggest that SARS-CoV-2 skews the antiviral response in favor of an NF-κB-driven inflammatory response, a hallmark of acute COVID-19 and for which Nsp2 should be considered an important contributor.

Efficient megakaryopoiesis and platelet production require phospholipid remodeling and PUFA uptake through CD36.

Lipids contribute to hematopoiesis and membrane properties and dynamics, however, little is known about the role of lipids in megakaryopoiesis. Here, a lipidomic analysis of megakaryocyte progenitors, megakaryocytes, and platelets revealed a unique lipidome progressively enriched in polyunsaturated fatty acid (PUFA)-containing phospholipids. In vitro, inhibition of both exogenous fatty acid functionalization and uptake and de novo lipogenesis impaired megakaryocyte differentiation and proplatelet production. In vivo, mice on a high saturated fatty acid diet had significantly lower platelet counts, which was prevented by eating a PUFA-enriched diet. Fatty acid uptake was largely dependent on CD36, and its deletion in mice resulted in thrombocytopenia. Moreover, patients with a CD36 loss-of-function mutation exhibited thrombocytopenia and increased bleeding. Our results suggest that fatty acid uptake and regulation is essential for megakaryocyte maturation and platelet production, and that changes in dietary fatty acids may be a novel and viable target to modulate platelet counts.

The role of platelets in immune-mediated inflammatory diseases.

Immune-mediated inflammatory diseases (IMIDs) are characterized by excessive and uncontrolled inflammation and thrombosis, both of which are responsible for organ damage, morbidity and death. Platelets have long been known for their role in primary haemostasis, but they are now also considered to be components of the immune system and to have a central role in the pathogenesis of IMIDs. In patients with IMIDs, platelets are activated by disease-specific factors, and their activation often reflects disease activity. Here we summarize the evidence showing that activated platelets have an active role in the pathogenesis and the progression of IMIDs. Activated platelets produce soluble factors and directly interact with immune cells, thereby promoting an inflammatory phenotype. Furthermore, platelets participate in tissue injury and promote abnormal tissue healing, leading to fibrosis. Targeting platelet activation and targeting the interaction of platelets with the immune system are novel and promising therapeutic strategies in IMIDs.

Lipidomic analysis of differently prepared platelet concentrates in additive solution during storage.

BACKGROUND: Structural and biochemical changes in stored platelets are influenced by collection and processing methods. Lesions may appear during platelet concentrate storage, some of which may be involved in adverse transfusion reactions. The preparation and storage of platelet concentrates (PC) may modify and even damage the lipid mediator content. The aim of this study was to investigate the lipidomic profile identified in the supernatants of PCs according to processing and storage conditions, both after leukocyte filtration and contained in platelet additive solution (PAS), comparing single donor apheresis (SDA) products with pooled buffy coat (BC) products. MATERIALS AND METHODS: We investigated the accumulation of various lipid mediators including lysophospholipids (LP) and eicosanoids in SDA and BC products stored for 0-5 days. All products were processed following French Blood Establishment (EFS) procedures in accordance with EDQM/GTS European Standards. Both SDA and BC were leukocyte reduced and conserved in 35% autologous donor plasma and 65% platelet additive solution. Lipidomic analysis was performed on PC supernatants using LS/MS spectrometry. RESULTS: Our data demonstrate that lysophosphatidylcholine (LPC) levels were higher in BCs compared to SDAs, with no difference in lysophosphatidic acid (LPA) expression between the two preparation methods. Results for other eicosanoids showed greater similarity; indeed, no clear pattern emerged from analysis of eicosanoids in terms of storage time and process. In general, we observed longitudinal lipid mediator modulation for both SDAs and BCs, particularly at later time points. DISCUSSION: The expression of LPC and some eicosanoids in BCs could be used as novel biomarkers of PC quality. Future studies are needed to explore their impact on adverse transfusion reactions.

The alarmin interleukin-1α triggers secondary degeneration through reactive astrocytes and endothelium after spinal cord injury.

Spinal cord injury (SCI) triggers neuroinflammation, and subsequently secondary degeneration and oligodendrocyte (OL) death. We report that the alarmin interleukin (IL)-1α is produced by damaged microglia after SCI. Intra-cisterna magna injection of IL-1α in mice rapidly induces neutrophil infiltration and OL death throughout the spinal cord, mimicking the injury cascade seen in SCI sites. These effects are abolished through co-treatment with the IL-1R1 antagonist anakinra, as well as in IL-1R1-knockout mice which demonstrate enhanced locomotor recovery after SCI. Conditional restoration of IL-1R1 expression in astrocytes or endothelial cells (ECs), but not in OLs or microglia, restores IL-1α-induced effects, while astrocyte- or EC-specific Il1r1 deletion reduces OL loss. Conditioned medium derived from IL-1α-stimulated astrocytes results in toxicity for OLs; further, IL-1α-stimulated astrocytes generate reactive oxygen species (ROS), and blocking ROS production in IL-1α-treated or SCI mice prevented OL loss. Thus, after SCI, microglia release IL-1α, inducing astrocyte- and EC-mediated OL degeneration.

The role of mitochondria in rheumatic diseases.

The mitochondrion is an intracellular organelle thought to originate from endosymbiosis between an ancestral eukaryotic cell and an α-proteobacterium. Mitochondria are the powerhouses of the cell, and can control several important processes within the cell, such as cell death. Conversely, dysregulation of mitochondria possibly contributes to the pathophysiology of several autoimmune diseases. Defects in mitochondria can be caused by mutations in the mitochondrial genome or by chronic exposure to pro-inflammatory cytokines, including type I interferons. Following the release of intact mitochondria or mitochondrial components into the cytosol or the extracellular space, the bacteria-like molecular motifs of mitochondria can elicit pro-inflammatory responses by the innate immune system. Moreover, antibodies can target mitochondria in autoimmune diseases, suggesting an interplay between the adaptive immune system and mitochondria. In this Review, we discuss the roles of mitochondria in rheumatic diseases such as systemic lupus erythematosus, antiphospholipid syndrome and rheumatoid arthritis. An understanding of the different contributions of mitochondria to distinct rheumatic diseases or manifestations could permit the development of novel therapeutic strategies and the use of mitochondria-derived biomarkers to inform pathogenesis.

Cytokines and Lipid Mediators of Inflammation in Lungs of SARS-CoV-2 Infected Mice.

Coronavirus disease 19 (COVID-19) is the clinical manifestation of severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2) infection. A hallmark of COVID-19 is a lung inflammation characterized by an abundant leukocyte infiltrate, elevated levels of cytokines/chemokines, lipid mediators of inflammation (LMI) and microthrombotic events. Animal models are useful for understanding the pathophysiological events leading to COVID-19. One such animal model is the K18-ACE2 transgenic mice. Despite their importance in inflammation, the study of LMI in lung of SARS-CoV-2 infected K18-ACE2 mice has yet to be studied to our knowledge. Using tandem mass spectrometry, the lung lipidome at different time points of infection was analyzed. Significantly increased LMI included N-oleoyl-serine, N-linoleoyl-glycine, N-oleoyl-alanine, 1/2-linoleoyl-glycerol, 1/2-docosahexaenoyl-glycerol and 12-hydroxy-eicosapenatenoic acid. The levels of prostaglandin (PG) E1, PGF2α, stearoyl-ethanolamide and linoleoyl-ethanolamide were found to be significantly reduced relative to mock-infected mice. Other LMI were present at similar levels (or undetected) in both uninfected and infected mouse lungs. In parallel to LMI measures, transcriptomic and cytokine/chemokine profiling were performed. Viral replication was robust with maximal lung viral loads detected on days 2-3 post-infection. Lung histology revealed leukocyte infiltration starting on day 3 post-infection, which correlated with the presence of high concentrations of several chemokines/cytokines. At early times post-infection, the plasma of infected mice contained highly elevated concentration of D-dimers suggestive of blood clot formation/dissolution. In support, the presence of blood clots in the lung vasculature was observed during infection. RNA-Seq analysis of lung tissues indicate that SARS-CoV-2 infection results in the progressive modulation of several hundred genes, including several inflammatory mediators and genes related to the interferons. Analysis of the lung lipidome indicated modest, yet significant modulation of a minority of lipids. In summary, our study suggests that SARS-CoV-2 infection in humans and mice share common features, such as elevated levels of chemokines in lungs, leukocyte infiltration and increased levels of circulating D-dimers. However, the K18-ACE2 mouse model highlight major differences in terms of LMI being produced in response to SARS-CoV-2 infection. The potential reasons and impact of these differences on the pathology and therapeutic strategies to be employed to treat severe COVID-19 are discussed.